This invention relates to the field of microfluidics-based automated chemistry.
Many analytical chemical problems involve the analysis of small amounts of a sample. The ability to do chemical reactions in small volumes of liquid is important in such cases as the reaction rate can decrease to commercially unacceptable levels if the concentration of the sample is too low. Additionally, for analytical devices used to monitor the results of such reactions, it is frequently important that the reaction product be in a small volume in order that a detectable concentration be present. The present invention offers a solution to the problem of doing chemical reactions in small volumes, to doing a succession of such reactions, and to doing them on an automated basis.
Although the present invention will be seen to have general chemical applicability, its application to biomedical research is of particular interest. Post-genome research will likely focus on understanding the cellular chemistry, circuitry and communications underlying life""s vital processes. Biomedical scientists, for instance, will aim to identify how genetic determinants of disease alter cellular physiology and response to agonists. Predictably, all this will involve biochemical analysis of larger numbers of samples containing ever lower concentrations of analyte. In most cases, analyses entail multistep procedures, including chemical and enzymatic reactions. Not only will automation prove essential in such cases, it must also be done in fully integrated instruments that incorporate the smallest possible reaction vessels and wetted surfaces. Progress towards this goal has come from nano-fabricated devices1-4 (xe2x80x9clab-on-a-chipxe2x80x9d), but severe limitations on sample volume may restrict the technology to fast, parallel analysis of abundant and/or amplifiable (e.g. by PCR) molecules. What""s needed is automation whereby trace analytes are processed in their entirety. We wanted to construct such a device, initially using chemical protein sequencing as a model system. Such chemistries have been replaced during recent years by mass spectrometry as a means to protein identification.5-8 Yet, the fact remains that chemical analysis can yield rather long stretches of easy to interpret sequence, including on intact proteins, with the caveat that current instruments are at least ten to twenty times less sensitive than most mass analyzers.
Traditionally, protein chemical sequencing is done by stepwise removal of amino acids from the N-teminal end, one at a time. In this method, the Edman degradation,9,10 phenyl isotliocyanate (PITC) is coupled to the alpha-amino group of the polypeptide to form a phenyl thiocarbamyl (PTC)-derivative; anhydrous acid causes selective release of the PTC-amino, leaving a truncated peptide chain. The resulting anilino thiazolinone amino acid (ATZ-aa) is converted with aqueous acid to a more stable phenyl thiohydantoin amino acid (PTH-aa) and identified. The latter step cannot be done in the presence of polypeptide as it would cause hydrolysis; thus, the ATZ-aa must be extracted. The procedure was partially automated, first by Edman in the xe2x80x98spinning cupxe2x80x99 version,11 and later in Laursen""s solid-phase sequencer.12 Conversion was initially done outside the machine, but later incorporated into the automated process by Wittman-Liebold.13 Until then, thin layer or gas chromatography were used for PTH-aa identification; Hunkapiller and Hood were among the first to routinely use reversed-phase high performance liquid chromatography (RP-HPLC) for this purpose.14 Around 1980, the gas-phase (GP) sequencer was developed by Hewick and coworkers.15 Here, wash-out of the peptide from the reaction vessel was prevented by non-covalent immobilization on a glass-fibre disc and by delivering polar liquids as vapors. The process has been further automated by coupling HPLC identification of PTH-aa""s xe2x80x98on-linexe2x80x99 with the sequencer; contents of the conversion flask are hereby directly transferred into the LC injector loop. Since then, progress has come from incremental improvements such as femtomole level HPLC detection,16 rigorous instrument optimization and maintenance routines,17 and the use of a smaller reaction cartridges.17-19 Combined with improved micro-preparation of polypeptides,20-22 chemical sequencing at the 1-3 picomole level is currently possible and extended sequencing runs with femtomole level signal have been reported.16,23,24 Additional modifications to the process have since been suggested that should allow true femtomole level sequencing. All relate to increased sensitivities of amino acid derivative detection. This can be accomplished by miniaturizing the HPLC-based PTH-aa detection system, or by producing modified Edman end-products of higher detect ability, or both.
Whereas femtomole level PTH-aa separations on microbore columns can be done,19,25 the use of capillary columns (300-micron ID) is more problematic, as injection volumes of over 0.5 xcexcL cause baseline disturbances.26 Complete injection (or even 10%) of the sample in a 0.5-xcexcL volume is not possible from any commercial automated sequencer. This also applies to capillary zone electrophoresis (CZE)-based amino acid-derivative detection systems.27 
Considerable effort has been expended to generate fluorescent amino acid derivatives as end-products of Edman chemistry. Fluorescent sequencing is especially appealing since the introduction of sub-attomole amino acid analysis by CZE with laser-induced fluorescence.27,28 Yet, again, loading volumes are in the nanoliter range, several orders of magnitude less than what is typically present in a sequencer flask. Another approach to xe2x80x98high-detect abilityxe2x80x99 is the generation of quaternary or tertiary amine thiohydantoin amino acid derivatives, analyzable by mass spectrometry at the low femtomole level.29,30 However, both methods never progressed beyond the RandD stage.
Development of any Edman based, femtomole-level technique requires satisfying two major criteria: (i) quantitative transfer, in the smallest possible volume, of amino acid derivatives to the site of analysis; and (ii) reducing chemical background that will impede any ultra-sensitive analytical technique. This can only be accomplished by further miniaturization of the chemistry.
We describe a microfluidics-based instrument, consisting of multiple rotary valves, capillary tubing and miniaturized reaction vessels, for the purpose of performing automated chemical and biochemical reactions on a very small scale. Close to 100% of the reaction end-products are available in a minimal volume (xe2x89xa65 xcexcL) inside a pressurized mirco-vial for subsequent analysis. This makes the system compatible with capillary HPLC and, in principle, with continuous flow nano-electrospray mass spectrometry. Total control of flow path combinations and directions, temperatures and gas pressures enables precise execution of complex biochemical laboratory procedures. Instrument performance was convincingly demonstrated by partially sequencing 100 femtomoles of an intact protein using classical Edman chemistry in combination with capillary-bore liquid chromatography. To our knowledge, this is the smallest amount of protein ever reported to be successfully analyzed in this way.
The invention is a chemical system and related processes that utilize small-volume rotary selector valves and small-volume rotary switching valves in combination under the control of a computer. The small rotary valves are particular well-suited to computer control. As a result, a single system can be programmed to do a variety of tasks by changing the program but leaving the system""s components substantially intact.
As a result, in a general aspect the invention is a system for carrying out one or more chemical reactions, said system comprising a rotary selector valve and a rotary switching valve, each valve under the control of a computer, the internal volumes of the selector and switching valves each being 1.5 xcexcl or less.
Specific aspects of the invention that are of particular interest are the selector valve-switching valve combination, a core system for regulating a sequence of reactions, a core system adapted for polymer sequence analysis, and processes that implement the systems. The core systems can, as indicated by the use of the word xe2x80x9ccomprisingxe2x80x9d in their description, be either used as stand-alone systems or be part of larger systems.
Selector Valve-Switching Valve Combination
An important aspect of the invention is a rotary valve combination comprising a rotary selector valve (with its plurality of peripheral ports) connected by its central common port to a peripheral port of a rotary switching valve. The connection is preferably accomplished by a conduit means (e.g., a tube or capillary). It is preferred that the conduit means have an internal volume between 0.5 xcexcL and 10 xcexcL.
The Rotary Selector Valves
It is preferred that the rotary selector valve comprise, in a first unit (e.g., the stator), a central common port and a circular array of a plurality of peripheral ports such that the main axis of the common port is the same is as the main axis through the circular array, and wherein said valve comprises, on a second unit, a radial connector channel, the first and second units being juxtaposed such that there is a single continuous selector channel formed by the common port, the connector channel, and a peripheral port, the selection of the peripheral port under control of the computer (the control preferentially effectuated by rotating the second unit), the internal volume of the continuous selector channel being the internal volume of the valve,
Preferably the two units are positioned in flat contact with each other, the flat contacting surface of the first unit against the flat contacting surface of the second unit. Contact between the two units is such that both the common port and a peripheral port meet the connector channel. This is optimally done by constructing the connector channel as a groove on the contacting surface of the second unit. It is preferred, but not required, that the common port act as an output port and that the connected peripheral port act as an input port, but the reverse relationship between the ports is also possible. The total internal volume (central port plus connector channel plus peripheral port) is preferably 1.5 xcexcL or less (more preferably in the range 0.04 xcexcL to 1.5 xcexcL, most preferably 0.1 xcexcL to 0.5 xcexcL).
The Rotary Switching Valves
It is preferred that the rotary switching valve comprise a circular array of three or more peripheral ports in a first unit (e.g., the stator) and a connector channel in a second unit, the two units being juxtaposed such that there is a continuous switching channel formed by a first peripheral port, the connector channel, and a second peripheral port, the selection of two peripheral ports being under the control of the computer (the control preferentially effectuated by rotating the second unit), the internal volume of the continuous switching channel being the internal volume of the valve.
Preferably the two units are positioned in flat contact with each other, the flat contacting surface of the first unit against the flat contacting surface of the second unit. Contact between the two units is such that two peripheral ports meet the connector channel. This is optimally done by constructing the connector channel as a groove on the contacting surface of the second unit. (The groove can be straight or curved). The total internal volume (central port plus connector channel plus peripheral port) is preferably 1.5 xcexcL or less (more preferably, in the range 0.04 xcexcL to 1.5 xcexcL, most preferably 0.1 xcexcL to 0.5 xcexcL).
System For Regulating a Sequence of Reactions
In a general aspect the invention is a computerized system for regulating complex reaction sequences in small volumes of solution, said system comprising:
a reaction vessel (for holding a volume of fluid in which a reaction takes place);
a first and second reagent vessel (for holding fluid reagents), said vessels each connected to a gas source,
a rotary valve combination connected to receive fluid from said reagent vessels and to deliver fluid to said reaction vessel, said combination optionally with other receiving-delivery capabilities, said rotary valve combination comprising a multi-position rotary selector valve connected to a rotary switching valve;
a computer connected to said valve combination so that said computer controls both whether the switching valve is configured to allow or to not allow fluid flow to the reaction vessel and whether the selector valve is configured for input from the first or second reagent vessel; and
wherein the internal volume of each of said valves along the path of fluid flow, is 1.5 xcexcL or less, (preferably in the range 0.04 xcexcL to 1.5 xcexcL, more preferably 0.1 xcexcL to 0.5 xcexcL).
Each gas source is selected from a group of one or more gas sources each comprising a gas under pressure, said gas optionally differing from source to source. Any gas or gas combination that is not chemically reactive with a fluid or other reagent in the system can be used.
System Adapted for Polymer Sequence Analysis
In another aspect, the selector valve of the above-noted system is a first selector valve and the switching valve thereof is a first switching valve, and the system further comprises:
a second rotary selector valve connected to a second rotary switching valve;
a third rotary switching valve;
a conversion vessel connected to receive output from said third rotary switching valve,
a restraining means for restraining unprocessed polymer in the reaction vessel;
third and fourth reagent vessels connected to provide fluid to said conversion vessel;
wherein said second rotary valve, second switching valve, and third switching valve are each under the control of the computer and each have an internal volume of 1.5 xcexcl or less (preferably 0.04 xcexcL to 1.5 xcexcL, more preferably 0.1 xcexcL to 0.5 xcexcL).
In particular embodiments, the system further comprises one or more of the following:
one or more wash solution vessels (each for holding a volume of fluid, preferably a wash solution), each said vessel connected to a gas source, each said vessel providing optional input (directly or via other elements of the system) via a selector valve to the reaction vessel and/or conversion vessel; and
a switching valve, under the control of the computer, for controlling fluid flow to an analytical device (such as an HPLC column or electrospray ionization mass spectrometer) from the conversion flask.
Reaction Vessel and Conversion Vessel
It is preferred that, when the first and second fluid reagents are in the reaction vessel, the volume of fluid in the reaction vessel is in the range of 0.5 xcexcL to 10 xcexcL (preferably 1 xcexcL to 5 xcexcL).
It is preferred that a volume of fluid in the conversion vessel be in the range 1 xcexcL to 150 xcexcL (preferably 3 xcexcL to 50 xcexcL, most preferably 3 xcexcL to 15 xcexcL).
Selector Valve Cascade
An aspect of the invention is a rotary selector valve cascade in which a plurality of peripheral ports of a first selector valve are each connected to the central common port of one of plurality of rotary selector valves that each comprise a plurality of peripheral ports, the total internal volume of each valve being 1.5 xcexcL or less (more preferably in the range of 0.4 xcexcL to 1.5 xcexcL, most preferably 0.1 xcexcL to 0.5 xcexcL).
Process Aspect of the Invention
In another general aspect, the invention is a reaction process under the control of a computer said process comprising the steps of:
1) Delivering, from a reagent vessel under pressure from a gas source, a volume of a reagent via a delivery line to a reaction vessel wherein the total volume delivered for purposes of a reaction in the reaction vessel is in the range 0.5 xcexcL to 10 xcexcL, preferably 1 xcexcL to 5 xcexcL, and wherein flow through the delivery line is under the control of a computer-controlled rotary valve combination; Preferred is the process with an additional steps (2) and (3):
2) Washing said valve combination and delivery line with a wash-solvent; and
3) Repeating step (1) optionally with a reagent, reagent vessel and/or gas source different from that used in step (1).
Process Aspect of the Invention Used to Sequence Polymers
In an aspect of the invention adapted for sequencing a polymer, the process comprises the steps of:
(1) Placing a polymer (preferably in the range 5 to 500 femtomoles, more preferably 20 to 250 femtomoles, most preferably 50 to 200 femtomoles) in a reaction vessel, said polymer restrained in said vessel, the restraining preferably accomplished by adsorption of the polymer to a solid support;
(2) Delivering to said reaction vessel, from a first reagent vessel under pressure from a gas source, a volume of a first fluid, said fluid comprising a first reagent, which reagent will react with a terminal monomer on the polymer;
(3) Delivering to said reaction vessel, from a second reagent vessel under pressure from a gas source, a volume of a second fluid, which fluid (the fluid can be a solvent or a solution of a solute in a solvent) will cause a terminal monomer-reagent moiety to be cleaved from the polymer;
(4) causing all or part of the fluid in the reaction flask to be transferred under gas pressure via a delivery line to a conversion flask while allowing the polymer, less the terminal monomer, to remain in the reaction vessel;
(5) Prior or after step (4), delivering from a third reagent vessel under pressure from a gas source a volume of a third fluid such that the combination of said fluid and the fluid transferred in step (4) to the reaction flask will cause the terminal monomer-reagent moiety to be cleaved to create a terminal monomer free of covalently bound reagent;
(6) Transferring under gas pressure from a gas source said terminal monomer created in step (5) to an analytical device that will identify the nature of the terminal monomer.
It is preferred that each volume delivered in steps (2), (3), (4), (5), is in the range 0.2 xcexcL to 10 xcexcL, (preferably 0.5 xcexcL to 5 xcexcL). Independently, it is preferred that the volume delivered in step (6) is in the range 1 xcexcL to 10 xcexcL, (preferably 2 xcexcL to 5 xcexcL).
In a preferred embodiment of the process adapted for sequencing a polymer, the process comprising steps (1)-(6), is performed a plurality of times.
Air-Free Reaction System
In preferred embodiments of the processes, the reaction vessel and the conversion vessel are kept free of air and oxygen. The ability to do this effectively is an advantage of the present invention.
Sequential Treatment with Acid and Base
In particular embodiments of the processes, the process comprises sequentially delivering acid, organic solvent, and base to the reaction vessel. The ability to do this effectively is an advantage of the present invention.
Applications of the Invention
Examples of such applications are:
1) automated reactor for solution chemistry;
2) automated reactor for liquid xe2x80x9cflow-throughxe2x80x9d chemistry;
3) automated reactor for gas-phase chemistry;
4) automated sample preparation, modification, and reaction prior to electrospray ionization mass spectrometry;
5) automated enzymatic digestion of proteins, nucleic acids and complex carbohydrates;
6) thermostatic control for sample preparation, modification and reaction (including the above uses);
7) automatic precision volume metering utilizing loops with volume range 0.1 microliter and above;
8) automated selection of samples for any use described above.
Specific Polymer Applications
In one set of embodiments, the system is adopted for processing a polymer in step with fashion, one monomer at a time.
Example of such processing are:
1) the determination of the amino acid sequence of a polypeptide;
2) the determination of the base sequence of a nucleic acid; and
3) the determination of the sugar sequence of a polysaccharide;